Epithelial barrier dysfunction contributes to progression of intestinal and systemic disease. However, there is a fundamental gap that separates clinicopathologic significance from molecular understanding of the mechanisms responsible for barrier regulation. The major components of the tight junction, which forms the paracellular barrier, have been identified over the past two decades and fall into three major groups; scaffold proteins, e.g. ZO-1; transmembrane structural proteins, e.g. occludin; and pore-forming proteins, e.g. claudins. How these proteins interact to regulate the barrier is incompletely understood. Thus, tight junction biology is at a crossroads that requires a transition from protein discovery to identification of essential regulatory mechanisms. These must be considered in the context of distinct components of paracellular permeability that define flux of either large solutes or small ions and water. We have recently shown that these aspects of barrier function are differentially regulated by the pathologically-relevant cytokines TNF and IL-13, respectively. The long term goal of these studies is to understand tight junction structure and regulation in molecular terms and to leverage this knowledge to develop approaches to modulate specific barrier components and improve health. The objective of this application is to define the interactions among tight junction components that regulate dynamic protein behavior and barrier function using newly-developed in vitro and in vivo approaches. Our central hypothesis is that the interactions responsible for tight junction protein anchoring and trafficking are the primary determinants of paracellular barrier function. This hypothesis has been formulated on the basis of strong preliminary data produced with support of the current funding cycle. The rationale for this project is that it will provide unprecedented insight into the molecular interactions that regulate barrier function and, in turn, will allow manipulation of these processes for therapeutic benefit. The hypothesis will be tested via specific aims: 1) To define the contributions of specific ZO-1 domains to trafficking, anchoring, and tight junction barrier regulation; 2) To determine the structural elements and phosphorylation events that regulate occludin trafficking, interprotein interactions, and in vitro and in vivo barrier function; and 3) To identify the structural elements and functional interactions by which claudin domains regulate pore assembly and opening. While each aim focuses on a critical tight junction protein or protein family, interactions among these will allow integration and development of a unified model of tight junction structure and regulation. The proposal is innovative because it explores the novel idea that dynamic regulation of protein interactions controls barrier function, which signals a major shift in our understanding of tight junction biology. The proposed research is significant because it will enhance our understanding of barrier dysfunction and link specific mechanisms of barrier loss to disease. The concepts and tools developed will make it possible to develop agents that target distinct barrier components and, ultimately, to treat diseases of epithelial and endothelial barriers.